Distraction osteogenesis (DO) is a widely used orthopedic treatment for the correction of limb length discrepancies, congenital deformities, non-unions, and the regeneration of large amounts of bone that have been lost due to trauma. It is one of the most dramatic applications of mechanical stimulation as a means of inducing regeneration of up to 20% of the length of a long bone. While DO procedures are used clinically and the surgical techniques have been refined, the basic mechanisms by which this procedure promotes new bone formation are not well understood. One of the primary descriptive features of distraction osteogenesis is that it induces new bone formation through an intramembranous processes devoid of extensive amounts of cartilage. However, the most intriguing observation is that the regeneration of abundant amounts of bone is accompanied by robust vascularity of the repair tissue throughout the regenerative process. The hypothesis of this proposal is that bone regeneration during distraction osteogenesis is driven by an angiogenic process. A component of this hypothesis is that the angiogenic events that accompany the regenerative process in distraction osteogenesis are the primary regulatory mechanisms that direct bone formation in the absence of chondrogenesis. In order to test this hypothesis and examine genetic models that will define the molecular mechanisms regulating bone formation, we have developed a murine model of DO. Initial studies will define the temporal and spatial expression of the angiogenic and bone morphogenetic signals that drive new bone formation during distraction osteogenesis and compare the expression of these signals to those that drive the endochondral repair processes that occur during fracture healing (these will be elucidated as part of Project 4). This will be followed with experiments in which we specifically block vascular endothelial growth factor (VEGF)-mediated angiogenesis using selective blocking antibodies to two different receptors of VEGF (Flk- 1 and Flt- 1), and functionally test the degree to which VEGF signals are mediators of both angiogenesis and/or new bone formation during distraction angiogenesis. We will test, in both in vivo and in vitro experiments, the relationship of BMP-2 stimulated differentiation to the expression of VEGF(s) and the inverse effects of VEGF on the expression of BMP(s). We will then determine how selective gain or loss of BMP activity during DO affects angiogenesis and how these alterations affect the formation of bone in this system.